1. Field of the Invention
The present invention relates to polynucleotide molecules (e.g., DNA, RNA, etc.). More particularly, the invention relates to antisense oligonucleotides useful for prevention, amelioration, and treatment of wheeze and other diseases related to pulmonary inflammation, pulmonary dysfunction, and airway hyperresponsiveness. In particular, the invention relates to antisense oligonucleotides useful for suppressing IL-4Rα-mediated signaling during a critical developmental period, thereby preventing, ameliorating, and treating RSV-associated airway diseases.
2. Description of Related Art
Asthma is epidemic among industrialized countries (Cookson W O & Moffatt M F, Science 1997; 275:41-42). Although molecular analyses have suggested a genetic basis for this disease state (se e.g., Daniels S E, et al. Nature 1996; 383:247-50; Van Eerdewegh P, et al. Nature 2002; 418:426-30), environmental factors are partly responsible. Hypotheses for the proliferation of asthma have focused on exacerbating factors such as air pollution (McBride D E, et al. Am J Respir Crit Care Med. 1994; 149:1192-97), early exposure to “trigger” antigens (e.g., dust mites, cockroaches) (O'Byrne P M. J Allergy Clin Immunol. 1988; 81:119-27), tobacco/chemical exposure (Flodin U, et al. Epidemiology 1995; 6:503-505), and increased amount of time spent outdoors versus indoors (Platts-Mills T A, et al. Curr Opin Immunol. 1998; 10:634-39). Moreover, the increased incidence of respiratory infections (e.g., respiratory syncytial virus (RSV), rhinovirus, and parainfluenza) associated with greater numbers of people living in high-density urban environments has also been proposed as a predisposing factor in the rising prevalence of asthma (Wang S Z & Forsyth K D. Clin Exper Allergy. 1998; 28:927-35). Despite intense study of patient populations, the unique circumstances that dictate why one person's immune responses lead to asthma when others' do not are still obscure.
Asthma is a respiratory disorder characterized by recurring episodes of paroxysmal dyspnea (sudden shortness of breath), wheezing on expiration due to constriction of bronchi, coughing, and viscous mucoid bronchial secretions (Mosby's Medical and Nursing Dictionary, 1990). Wheeze is a form of rhonchus (abnormal sounds heard upon auscultation of a respiratory airway obstructed by, e.g., thick secretions), characterized by a high-pitched musical quality and caused by a high-velocity flow of air through a narrowed airway (Id). Wheezes may be heard both during inspiration and expiration, and are associated with asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), pulmonary edema, and respiratory infections, among other things. Although the etiology and symptoms of asthma are highly variable among patients, three common characteristic features of asthma exist: reversible variable airflow limitations; specific airway histopathologies due to airway inflammation and damage; and airway hyperresponsiveness (AHR, the development of bronchoconstriction in response to nonspecific inflammatory stimuli) (Bochner B S, et al. Annu Rev Immunol. 1994; 12:295-35). The onset and progression of allergic asthma is accompanied by a complex series of overlapping and concurrent inflammatory responses in the lung orchestrated by CD4+ Th2 lymphocytes (Type 2 T helper cells expressing CD4+). (Gavett S H, et al. Am J Respir Cell Mol Biol. 1994; 10:587-93. Kon O M & Kay A B. Int Arch Allergy Immunol. 1999; 118:133-35. Robinson D, et al. J Allergy Clin Immunol. 1993; 92:313-24. Walker C, et al. Am Rev Respir Dis. 1992; 146(1):109-15. Kay A B. Ciba Found Symp. 1997; 206:56-67.) These responses include T cell mediated help of antigen-specific immunoglobulin production particularly IgG1 and IgE by B cells (DeKruyff R H, et al. Semin Immunol. 1993; 5:421-30. Jenmalm M C, et al. Pediatr Allergy Immunol. 1999; 10:168-77.), expression of Th2 proinflammatory cytokines (e.g. IL-4, -5, -9, and -13) (Hoppler S & Bienz M. Cell. 1994; 76:689-702. Host A H, et al. Ugeskr Laeger [Danish]. 1993; 155:3978-81. Till S, et al. Immunology 1997; 9153-57.), and the activation of stromal and epithelial cells leading to the release of chemokines that initiate and perpetuate lung inflammatory reactions (Levine S J. J Investig Med. 1995; 43:241-49.). Asthma-associated pulmonary inflammation is also characterized by cellular infiltrates, which are believed to be involved subsequently in histopathologies and are also thought to be the underlying cause(s) of the accompanying airway obstruction and lung dysfunction.
For many years, it was thought that “reversible” airflow obstruction meant that asthma was also reversible. Thus, it was anticipated that childhood asthma was a self-limiting disorder that the child would “outgrow.” However, recent evidence suggests that repeated injury to the lung results in structural airway changes that—under some conditions—are irreversible. It is now well recognized that the majority of children with moderate to severe symptoms continue to wheeze and have reduced lung function values as adults. Even though they may consider themselves “symptom-free,” they continue to show reduced lung function and increased bronchial reactivity to both specific and non-specific stimuli (Pedersen S, “Asthma in Children,” In: Asthma Basic Mechanisms and Clinical Management (Peter J. Barnes, Ian W. Rodger, and Neil C Thompson, eds., Academic Press 1998), 3d ed., pp. 859-902.). The exact reason(s) for the long-term decline in lung function is uncertain, but pulmonary remodeling is a likely explanation. Supporting evidence comes from a study in which rats were repeatedly exposed to aerosolized ovalbumin (OVA) (Palmans E, et al. Am J Respir Crit Care Med. 2000; 161:627-35.). This resulted in AHR to carbachol, which was accompanied by structural changes/remodeling of the airways, including goblet cell hyperplasia, increased proliferation of airway epithelium, increased deposition of fibronectin, and increased thickness of the interstitial matrix (Palmans E, 2000).
Interestingly, human studies have demonstrated that airway remodeling events associated with asthma begin early in life, and in some infants are observable at the pathological level prior to the clinical onset of asthma symptoms (Warner J A. J Allergy Clin Immunol. 2000; 105:951-59. Group TCAMPR. N Engl J Med. 2000:343; 1054-63.). The immature lung is saccular (sack-like) in structure, and has a limited gas-exchange capability. Maturation into a mature lung with a large internal surface area capable of highly efficient gas exchange requires thinning of the alveolar walls, extensive subdivision of saccular lung into alveoli, and growth of the pulmonary capillary network (Ad hoc Statement Committee, American Thoracic Society. Am J Respir Crit Care Med. 2004; 170:319-43.). In humans, this maturation process begins at 36 weeks of gestation. Only 15% of the alveoli have formed at birth, and maturation continues into the third year of life (Dunnill M. Thorax. 1962; 17:329-33. Burn P, “Structural aspects of prenatal and postnatal development and growth of the lung,” In: Lung Growth and Development (John A. McDonald ed., Informa Healthcare 1997), 1st ed., pp. 1-35. Merkus P, et al. Pediatric Pulmonology. 1996; 21:383-97. Meyrick B & Reid L, “Ultrastructure of alveolar lining and its development,” In: Development of the Lung: Lung Biology in Health and Disease Series (W. A Hodson ed., Marcel Dekker 1977), pp. 135-214.). Rodents are also born with the lung in saccular stage, with alveolarization and wall thinning occurring postnatally. At birth, the immature murine lung lacks alveoli, alveolar ducts, and respiratory bronchioles. Alveolarization in the rat occurs on portpartum days 4-7, and respiratory bronchioles are found 10 days after birth (Burn P. Anat Rec. 1974; 180:77-98.). Interestingly, increased oxidative stress due to mechanical lung ventilation in pre-term human infants causes extensive alveolar fibroproliferation, smooth muscle hyperplasia, and inhibition of distal lung formation, and also leads to long-term pulmonary dysfunction persisting into adulthood (Northway W, et al. N Engl J Med. 1967; 276:357-68. Kurzner S I, et al. J. Pediatr. 1988; 112:73-80.). These data suggest that infant lung tissue responds to external environmental factors that can influence pulmonary patterning, extent of lung growth, and long-term physiologic function.
Three broad influences are currently believed to be the most important factors in the development of asthma: genetics; environmental factors (i.e., exposure to allergens or pathogens); and interactions between these factors and the developing immune/pulmonary system in early life. As a fetus, and shortly after birth, the immune system is prone to Th2 responses (Adkins B. Int Rev Immunol. 2000; 19(2-3):157-71.). In the fetus this Th2 bias is thought to protect both the mother from cytotoxic Th1 (Type 1 T helper cell) fetal responses and the fetus from maternal rejection (Wegmann T G, et al. Immunol Today. 1993; 14:353-56.). After birth, the immune system begins to mature in an age- and exposure-dependent manner. In humans, the Th2 cytokine profile persists throughout the first year of life and is accompanied by a relative eosinophilia (Prescott S L, et al. Lancet. 1999; 353:196-200. Bruce M. Camitta, “The anemias,” In: Nelson Textbook of Pediatrics (Ricard E. Behrman, Robert M. Kliegman, and Ann M. Arvin, eds., W.B. Saunders 1996) 15th ed., pp. 1379.). In mice, the Th2 cytokine profile persists until approximately three weeks of age (Becnel D, et al. Respir Res. 2005; 6:122. You D, et al. Respir Res. 2006; 7:107.).
One particular environmental factor—respiratory syncytial virus (RSV), a member of a subgroup of myxoviruses—is the most common cause of bronchiolitis (acute viral infection of the lower respiratory tract) and pneumonia (acute inflammation of the lungs) in humans during infancy. Usually, symptoms begin with fever, runny nose, cough, and sometimes wheezing. During their first RSV infection, approximately 25% to 40% of human infants present signs of bronchiolitis or pneumonia, and they usually recover within 8 to 15 days. Approximately 0.5% to 2% of RSV-infected children require hospitalization, and the majority of these are under 6 months of age. Most human children have serologic evidence of RSV infection by 2 years of age (Glezen W P, et al. Am J Dis Child. 1986; 140:543-6.). RSV may cause repeated infections throughout one's life, with community-wide infections usually occurring in late fall, winter, or early spring months. A diagnosis of RSV infection may be made by detection of viral antigens, viral mRNA, or a rise in serum antibodies, by isolation of the virus, or by a combination of these strategies. Most commonly, antigen detection assays are employed. Moreover, infections of cattle and goats with bovine RSV and of sheep with ovine RSV are widespread, and produce significant economic losses (Mallipeddi S K & Samal S K. J Gen Vir. 1993; 74:2787-91.). Although the development of vaccines against RSV is a pressing research priority, they are either unavailable or present serious drawbacks.
Several retrospective and prospective human studies have suggested a link between RSV lower respiratory tract infections during infancy and later development of asthma (Sims D G, et al. Br Med J. 1978; 1:11-14. Pullan C R & Hey E N. Br Med J (Clin Res Ed). 1982; 284:1665-69. McConnochie K M & Roghmann K J. Pediatrics. 1984; 74:1-10. Mok J Y & Simpson H. Arch Dis Child. 1984; 59:306-9. Murray M, et al. Arch Dis Child 1992; 67:482-7. Noble V, et al. Arch Dis Child. 1997; 76:315-9. Stein R T, et al. Lancet. 1999; 354:541-5. Sigurs N, et al. Am J Respir Crit Care Med 2005; 171:137-41. Piippo-Savolainen E, et al. Allegy Asthma Proc. 2007; 28:163-69. Openshaw P M J. Clin Exp Immunol. 2003; 131:197-198.). Two ongoing longitudinal studies clearly demonstrate that RSV in early life does increase the risk of wheeze (and perhaps asthma) in later childhood (Stein R T, et al., 1999. Sigurs N, et al., 2005.).
In one study, 43% of children diagnosed with severe RSV bronchiolitis as infants still experienced asthma or wheeze at 13 years of age, compared to only 8% of control patients (Sigurs N, et al., 2005.). Interestingly, 50% of children who had RSV bronchiolitis also tested positive to aeroallergens, versus 28% of controls (Id.). These results suggest that severe RSV infection during infancy predisposes one not only to the development of asthma or wheeze, but also to the development of allergic disease (Id). Multivariant analysis demonstrated that the highest frequency of wheeze was observed when RSV bronchiolitis and a family history of atopy were present as risk factors (68% of the RSV group as compared to 34% of the control group) (Id). Results obtained from the Tucson Children's Respiratory Study found that children with even mild RSV infections were four times more likely to have recurrent, frequent wheeze by 6 years of age (Stein R T, et al., 1999.). By 13 years of age, though, the association between wheeze and RSV was no longer significant. This study also showed no relationship between RSV infection and positive skin reactivity tests to aeroallergens (Id). Some factors that may account for the lack of consistency between the prospective studies on the relationship between RSV and the development of asthma are: (i) recruitment of infants (only the sickest infants hospitalized); (ii) inaccuracy of RSV testing, which often requires multiple tests to achieve a positive result; (iii) reliance on parental answers to judge continued wheeze, as with the Tucson Study, (iv) differences in gestational ages of the children recruited; and (v) differences between RSV strains, which may produce different immunological and physiological responses. Cumulatively, the data suggest that RSV bronchiolitis in infancy is associated with an increased risk of wheeze, which may persist for several years and is not adequately explained by allergies or a family history of atopy.
Previous efforts at creating a human vaccine against RSV produced tragic consequences. In the mid-1960s, an experimental formalin-inactived RSV vaccine was developed and administered parenterally to infants between two and seven months of age (Kim H W, et al. Am J Epidemiol. 1969; 89:422-434.). The vaccine caused a measurable serum-neutralizing antibody response, but when RSV became prevalent in the community (i.e., when those vaccinated were later infected naturally) 80% of those vaccinated required hospitalization for pneumonia and/or bronchiolitis. Two infants died (Id.). Post-mortem examinations revealed pneumonia and patchy atelectasis, while histologic analysis revealed peribronchiolar eosinophilia (Id.). Further studies in adult mice using formalin-inactivated RSV confirmed the above findings (Power U F, et al. J. Virol. 2001; 75:12421-30. Peebles R S Jr., et al. J Infect Dis. 2000; 182:671-77.). Numerous subsequent studies have demonstrated that RSV infection enhances Th2 cytokine responses and eosinophilic infiltration following allergen sensitization and challenge (Becnel D, et al., 2005. You D, et al., 2006. Barends M, et al. Clin Exp Allergy. 2002; 32:463-71. Peebles R S Jr., et al. J Med Virol. 1999; 57:186-92.).
A recent study by Culley, et al. presented the first evidence demonstrating that “infections in early life play an important role” in shaping the secondary response to antigen, and can lead to long-term consequences for the host (Culley F J, et al. J Exp Med. 2002; 196:1381-86.). Culley, et al. demonstrated that the age of initial infection with RSV played a significant role in the secondary response to rechallenge with RSV. As seen in FIG. 1, the immune response of mice initially infected with RSV between 1 and 7 days of age and rechallenged at 12 weeks of age was characterized by increased bronchoalveolar lavage (BAL) cellularity, including increased eosinophil and neutrophil cell numbers, and increased CD8+ and CD4+ T cell production of intracellular interleukin 4 (IL-4) (FIG. 1). In contrast, the immune response of mice initially infected at 4 weeks of age and rechallenged at 12 weeks of age was characterized by decreased eosinophil and neutrophil cell numbers, decreased CD4+ T cell production of intracellular IL-4, and increased CD4+ T cell production of intracellular interferon gamma (IFN-γ) (FIG. 1). The work of Culley et al. suggests that the pattern of inflammatory cell response in infants may be important during re-infection but does not suggest how pulmonary dysfunction or RSV-related asthma later in life may be prevented.
The development of Th2 immune response is critically affected by IL-4 and IL-13 (FIG. 3). IL-4 is critical for the commitment of T helper cells to the Th2 lineage (relative to ml) and for IgE isotype switching, while IL-13 plays a critical role in the pathogenesis of allergic diseases including the development of AHR, lung remodeling, and mucus hyperproduction. IL-4 is a ligand for both the IL-4 type I (IL-4Rα and IL-4Rγc) and type II (IL-4Rα and IL-13Rα1) receptor heterodimers, and IL-13 exerts its actions by binding to the type II IL-4R (FIG. 3). IL-4Rα (see, e.g., SEQ ID NO:1, H. sapiens IL-4Rα, Accession No. NM—000418.2) is a high-affinity receptor for IL-4, and binding of IL-4 to this receptor promotes heterodimerization with a second chain (e.g., IL-4Rγc). Heterodimerization of IL-4R, in turn, activates down-stream signaling through members of the Janus kinase family, leading eventually to activation of signal transducer and activator of transcription (Stat6) protein and expression of various IL-4 inducible genes. Although a second IL-13 receptor (IL-13Rα2) exists, it is currently thought to be a non-signaling “decoy” receptor.
Recently, Karras, et al. demonstrated that reduction of IL-4Rα in the lungs (using inhaled antisense oligonucleotides against IL-4Rα) was sufficient to inhibit airway hyperresponsiveness and inflammation in an adult model of allergen-induced asthma (Karras J G, et al. Am J Respir Cell Mol. Bid. 2007; 36:276-85.). The findings of Karras, et al. support the use of inhaled IL-4Rα antisense oligonucleotides as a therapy for preexisting asthma and asthma exacerbations by essentially blocking Th2 effector cell function. In contrast, the present invention focuses on the use of IL-4R antisense oligonucleotides in the blocking the initiation of Th2 cellular differentiation and effector function in response to infant RSV infection, thereby inhibiting the pathophysiologic sequelae (e.g. persistent airway dysfunction and Th2 inflammatory responses upon subsequent exposure to RSV) that it initiates.
The technical problem underlying the present invention was therefore to overcome these prior art difficulties by inhibiting IL-4Rα in infants around the time that the T helper cell response is being influenced by RSV infection to develop as a Th2 response. The solution to this technical problem is provided by the embodiments characterized in the claims.